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DC-PNM-PTX improves the pharmacokinetics of PTX

Materials and Methods

BALB/c mice received a single intravenous injection of drug-free telodendrimer control, free PTX or DC-PNM at the PTX dose of 10 mg/kg body weight. 14C-labeled PTX was used in this study to allow the PTX concentration to be measured by liquid scintillation count. Blood was collected at different time points (0, 1, 3, 5, 15 and 30 minutes, 1, 2, 4, 8 and 24 hours post injection) in ethylenediaminetetraacetic acid (EDTA) tubes, and plasma was separated by centrifugation and kept at -20℃ until analysis. Five microliters of plasma of each specimen were used for scintillation count to determine PTX. The parameter profiles of PK studies were calculated and analyzed by using Kinetica Trial Version 5.0 (Adept Scientific Ltd, London, UK).

At 4, 24 and 48 hr post-injection, major organs (heart, liver, spleen, lung and kidney) were harvested. Tissues (100 mg) were mixed with 900 µl extraction buffer, and homogenized using POLYTRON® PT 10–35 homogenizer (Kinematica, AG, Littau/Lucerne, Switzerland). The samples were centrifuged at 3000 rpm for 5 min after mixing, and the supernatant was used to determine PTX concentration. Sample recovery standard curves for each tissue were generated by mixing a range of free PTX concentrations with each homogenized tissue type followed by the same extraction and quantification procedure. The biodistribution data was expressed as the % injected dose per gram tissue.

Results

Both free PTX and DC-PNM-PTX had a characteristic first phase of rapid tissue distribution and decline of plasma PTX concentration. After the initial rapid phase, the blood clearance of PTX continued to be cleared at a much slower pace. However, free PTX was much more rapidly cleared from the plasma with a half-life in plasma of 526.9 +/- 182.9 min (T1/2β), compared to 929.5 ± 134.8 min (p=0.037) with DC-PNM-PTX. Our analysis did not distinguish free PTX from PTX in micelles since PTX was extracted during analysis. However, considering the same experimental condition between these two groups, the prolonged half-life of PTX in DC-PNM was most likely secondary to the micellar formulation that trapped DNR in blood circulation.

DC-PPM (10 mg/kg) in Balb/C mice(blood)

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Free PTX (10 mg/kg)in Balb/C mice(blood)

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T1/2: Free PTX: 526.91±182.80

DC-PTX: 929.55±134.79

Note: Cmax, the peak plasma concentration of a drug AUC: Area under the concentration-time curve AUC extra: Area under the concentration-time curve extrapolated from t0 to ∞ in % of the total AUC AUC tot: area under the curve AUCextra%: Area under the concentration-time AUMC : Area under the first moment of the concentration-time curve AUMCextra: Area under the first moment of the concentration-time curve extrapolated from tz to ∞ in % of the total AUMC

AUMC tot, total area under the moment curve Thalf (min): elimination half-life P values: comparison of free PTX and DC-PTX

Drugs No. Cmax AUC AUCextra AUCtot %AUCextra AUMC AUMCextra AUMCtot Thalf(min)

Free PTX 1 96000 8150000 1020000 9170000 11.13 3100000000 2900000000 5900000000 734.159 2 100100 7880000 452400 8340000 5.42 2600000000 1100000000 3700000000 457.971 3 80800 7490000 409572 7900000 5.18 2600000000 900000000 3600000000 388.608

DC-PTX 4 8500 6260000 2130000 8390000 25.44 4900000000 6400000000 11300000000 881.137 5 9200 6230000 3100000 9320000 33.22 4500000000 10000000000 14700000000 1081.86 6 8900 5520000 2390000 7910000 30.21 3400000000 6300000000 9700000000 825.662

P values

0.0001 0.004 0.005 0.91 0.002 0.035 0.012 0.011 0.037

The pharmacokinetic parameters of free PTX and paclitaxel micellar formulations

The histopathological changes were investigated by H&E staining of different organs from mice administrated with PBS control, free PTX, and DC-PPM-PTX groups. The figure below showed that, in the liver section of free PTX and DC-PNM-PTX mouse, there are punctuate hepatocytes injury characterized individual cell lysis, apoptosis with nuclear irregularity, pyknosis, cytoplasmic vacuolization, microvesicular steatosis and occasional inflammatory reaction, but in the DC-PNM-PTX mouse the toxicity is much decreased. In the lung tissue, no specific epithelial damage is detected in spite some nonspecific alveolar filling is seen which could be due to processing changes and artifact.

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PBS Free PTX DC-PPM-PTX 40× 400× 40× 400× 40× 400×

We also studied the drug delivery to major organs at 4, 24 and 48 hours after dosing. In most organs, there was not much difference in PTX concentration at the corresponding time points between these two groups. One exception is that the 48-hour PTX concentration in spleen was much higher in the free PTX group than the DC-PNM-PTX group (254.7 +/-18.9 versus 34.4 +/- 7.3 µg/g, p<0.001). It is not known whether the difference in one organ at one time point is clinically significant. However, considering that we have so many comparisons (nine major organs with three comparisons in each organ), it is possible that this observed difference in one organ at one single time is due to a random event.

Biodistribution of PTX formulations

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Organs after a single dose (10 mg/kg)

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